Colorimetric determination of chlorides in serum



United States Patent Otfice 3,185,549 COLORIMETRIC DETERMINATION OFCHLORIDES IN SERUlVI Robert H. Hamilton, Philadelphia, Pa., assignor toHartman-Leddon Company, Inc., Philadelphia, Pa., a corporation ofDelaware No Drawing. Filed Aug. 27, 1962, Ser. No. 219,732 12 Claims.(Cl.;23--230) This invention relates to a reagent and method fordetermining the presence of small amounts of chloride in serum, thereagent and its method being suitable for making either visual orspectrophotometric comparisons in determining minute amounts ofchloride.

a A principal object of the present invention is to provide a method anda reagent useful for making accurate colorimetric determinations ofchloride in blood serum and without producing precipitates which wouldinterfere with or prevent the obtaining of meaningful test results. Inthis connection, it is a specific object to provide a method based onthe displacement of thiocyanate fromm-ercuric thiocyanate by chlorideions and the subsequent reaction of the liberated thiocyanate withferric ions to form the colored ferric thiocyanate complex, the colorproduced by such .a reaction being intensified by the presence ofperchloric acid without the attending precipitation of proteins by thatacid. Another object is to providea direct photometric method andreagent for determining the presence of chloride inserum in which thecolor development is substantially linear over therange to be tested.Other objects will appear as the specificationproceeds.

An important aspect of the present invention lies in the discovery thatthe presence of chloride in serum may be determined colorimetrically,without'the formation of a protein precipitate, if a measured quantityof urea is included in the testing solution. Further, it has been foundchlorides may be detected colorimetrically by treating a sample with atest solution of perchloric acid. containing mercuric thiocyanate andferric ions. The fundamental reactions that take place are as follows:

These reactions require the presence of a strong mineral acid such asperchloric acid. While various concentrations may be used, it has beenfound that the color of the ferric thiocyanateis greatly intensified thefinal perchlon'c acid concentrationis within the range of 35 to 45percent, the optimum concentration being approximately 41 percent. Theresults at acid concentrations below this range tend tobe erratic and athigher concentrations the color developed with serum tends to fade.Generally, perchloric acid concentrations in the lower range can betolerated if spectrophotometric measurements are to be made rather thanvisual comparisons. r 1

Ferric perchlorate is preferred as a source of ferric ions. Other ferricsalts can be used, however, as long as they do not introduce extraneousanions which will behave adversely in the strong acid medium.

Since mercuric thiocyanate is not readily soluble as such, it has beenfound desirable to add measured amounts of potassium thiocyanate andmercuric perchlorate to the acid solution, the two ingredients therebyproducing a known quantity of dissolved mercuric thiocyanate. Preftimesthe serum volume.

3,l85,54 Patented May 25, 1965 era-bly, a slight excess of mercuricperchlorate is provided s nce the presence of additional mercuric ionsserves to limit color development by requiring chloride ions in excessof the mercuric ions before a color change occurs. when a samplecontaining a sufiicient quantity of chloride 10118 is reacted with thetest solution so that the red color of ferric thio-cyanate appears, thesteep curve of color development with increasing amounts of chloridepermits an accurate chloride determination without reaching opticaldensities beyond the range of ordinary photometers.

In addition to the above ingredients, the test solution also containsurea and a small amount of mercuric chloride. Generally, the amount ofurea, when measured volumetrically as a 50 percent weight to weightaqueous solution, should be at least equal to times the volume of serumof the test sample. Preferably, the volume of the added urea solutionshould range between to 45 Since normal testing procedure involves 10milliliters of testing solution and 0.1 milliliter of serum, it has beenfound that a suitable testing solution may be prepared by usingapproximately 170 to 250 grams of undiluted urea for each liter of finalsolution.

meaningful colorimetric determinations.

An optimum amount of urea is found to be approximately 200 grams perliter of final solution or, when the urea is previously diluted with anequal part by weight of water, a volume of approximately 350 millilitersfor each liter of final solution.

The urea solubilizes the protein of the serum and prevents it from beingprecipitated by the perchloric acid of solution on standing.

final testing solution.

The excess mercuric ions added as mercuric perchlorate su'btract or binda fixed amount of the chloride to be determined. Removal of this fixedamount of chloride leaves a shorter range of chloride concentrations toreact with the mercuric thiocyanate, and thus permits use of the greatercolor intensity, with a greater change of absorbance per unit change ofchloride concentration. Photometric accuracy is thereby enhanced. Themicroequivalents of excess mercuric perchlorate added should be somewhatless than the minimum number of microequivalents of chloride expected inthe samples to be analyzed.

Mercuric chloride'in the testing solution improves linearity. The amountof mercuric chloride in the test solution is small and preferably rangesbetween 0.1 to 0.2 grams for each liter of the solution. Particularlyeffective results have been found where approximately 0.16 gram ofmercuric chloride is used in preparing the This is accomplished byadding 6.0 milliliters of 0.1 molar mercuric chloride to the otherreagents in 1 liter of the testing solution. Besides improving thelinearity, the amount of mercuric chloride can be used to adjust theabsorbance range produced after color development, in order that athickness of solution optimum for a specific photometer can be used.

In performing a test, an accurately measured quantity of serum, usually0.1000 milliliter, is mixed in a photometer tube with exactly 10milliliters of the reagent solution. Identical quantities of sodiumchloride standards (80, 100, meq. per liter) are added and mixed withthe reagent solution in three photometer tubes, each tube containingexactly 10 milliliters of the color solution. The tubes are stopperedand, after 20 to 30 minutes, are read in a photometer at a convenientwave length between 430 to 550 millimicrons, such as 515 millimicrons.After the absorbance of the ferric thiocyanate One liter of a colorreagent or testing solution embodying the present invention may beprepared from the following ingredients:

Ml. Urea, 50% (w./W.) 350 HClO 60% 600 Fe(ClO approx. 0.5 g./ml. 10.0KSCN, 0.4 M 12.5 Hg(ClO 0.2 M; add to an exact colorless endpoint;requires about 12.5 Hg(ClO 0.02 M 15.0 Hgcl 0.1 M 6.0

To prepare this solution, 500 g. of urea are dissolved in 500 g. (501.5ml.) of water, with warming to 3540. Total volume is about 875 ml. Ifthis solution looks dirty, it can be clarified by mixing it with 1 ml.of ZnSO -7H O, followed by 1 ml. of approximately 0.5 N NaOI-I. Aflocculation which appears may then be filtered out through acoarse-porosity sintered glass filter with washed asbestos fibers(analytical grade). The resulting solution is clear and colorless.

The ferric perchlorate is prepared by dissolving in water g. of acommercial ferric perchlorate, non-yellow, ex-

cess perchlorate acid preparation having a salt content of about 50% byweight, and thereafter adjusting the volume to 25 ml.

The 0.2 M mercuric perchlorate is prepared by adding 10 ml. of 60%perchlorate acid to 4.33 g. of pure mercuric oxide (red), and thenwarming to dissolve. Thereafter, the solution is cooled and diluted to100 ml. with water. Further dilution of a portion of this solutionproduces the 0.02 M mercuric perchlorate reagent.

The potassium thiocyanate constituent is prepared by dissolving 3.89 g.of pure potassium thiocyanate in water and then diluting to 100 ml., andthe mercuric chloride is similarly prepared by dissolving 2.72 g. of thesalt in water and diluting to 100 ml.

After the initial photometric determinations are taken as describedabove, two drops (about 0.02 to 0.04 ml.) of 0.2 M Hg(ClO.;) solution isadded to each photometer tube and mixed with the solution containedtherein. All ferric thiocyanate color fades out in the presence of theadded mercuric ions. The optical density is again determined andrecorded as the final reading to give the blank correction resultingfrom chromogens not decolorized by mercuric ions.

While in the foregoing I have disclosed an embodiment of the presentinvention in considerable detail for purposes of illustration, it willbe understood by those skilled in the art that many of these details maybe varied Without departing from the spirit and scope of the invention.

I claim:

1. In the colorimetric determination of chloride in serum, the steps ofreacting chloride in serum with mercuteases ric thiocyanate in aperchloric acid solution containing ferric ions, and simultaneouslysolubilizing the proteins of said serum in said solution with urea toprevent impairment of colorimetric determinations by reason of proteinprecipitates without at the same time reducing color intensity.

2. In the colorimetric determination of chloride in serum, the steps ofreacting the chloride of serum with mercuric thiocyanate in a perchloricacid solution containing ferric ions, and simultaneously solubilizingthe proteins of said serum in said solution with a volume of 50 percentby weight aqueous urea at least equal to 25 times the volume of saidserum.

3. The method of claim 2 in which 30 to 45 parts by volume of 50 percentby weight aqueous urea are provided for each part by volume of serum.

4. The method of claim 2 in which said solution also contains a minorproportion of mercuric chloride.

5. In the colorimetric determination of chloride in serum, the steps ofcontacting serum with mercuric thiocyanate in a strong mineral acidsolution containing ferric ions, and simultaneously solubilizing theproteins of said serum in said solution with urea.

6. The method of claim 5 in which the volume of 50 percent by weight ofaqueous urea in said solution is at least equal to 25 times the volumeof serum.

7. The method of claim 5 in which 30 to 45 parts by volume of 50 percentby weight aqueous urea are provided for each part by volume of serum.

8. In the colorimetric determination of chloride in a test solution, thestep of reacting said chloride with mercuric thiocyanate in a strongperchloric acid solution containing ferric ions, the concentration ofsaid perchloric acid in the final solution being within the range of 35to 45 percent for intensifying the color of the ferric thiocyanatereaction product.

9. A color reagent for the colorimetric determination of chloride inserum, comprising a strong mineral acid solution of mercuric thiocyanateand a ferric salt, said solution also containing to 250 grams of ureadissolved in each liter of said solution.

10. The reagent of claim 9 in which approximately 200 grams of urea aredissolved in each liter of said so lution.

11. A color reagent for the colorimetric determination of chloride inserum, comprising a perchloric acid solution of mercuric thiocyanate anda ferric salt, said solution also containing 170 to 250 grams of ureadissolved in each liter of said solution.

12. The reagent of claim 11 in which approximately 200 grams of urea aredissolved in each liter of said solution.

References Cited by the Examiner UNITED STATES PATENTS 3,055,742 9/62Hamilton 23-230 OTHER REFERENCES Wardle: Increasing Use of Urea inTextile Manufacturing, Textile Colorist, 62, Sept. 15, 1940, pages 629-630.

Practical Physiological Chemistry, Hawk et al., 12th ed., 1947, pages573-577 relied on.

MORRIS O. WOLK, Primary Examiner.

JAMES H. TAYMAN, JR., Examiner,

1. IN THE COLORIMETRIC DETERMINATION OF CHLORIDE IN SERUM, THE STEPS OFREACTING CHLORIDE IN SERUM WITH MERCURIC THIOCYANATE IN A PERCHLORICACID SOLUTION CONTAINING FERRIC IONS, AND SIMULATANEOUSLY SOLUBILIZINGTHE PROTEINS OF SAID SERUM IN SAID SOLUTION WITH UREA TO PREVENTIMPAIRMENT OF COLORIMETRIC DETERMINATIONS BY REASON OF PROTEINPRECIPITATES WITHOUT AT THE SAME TIME REDUCING COLOR INTENSITY.